Nucleic acid polymerases are an important class of compounds that enzymatically link (polymerize) nucleotides to form larger polynucleotide chains (e.g., DNA or RNA strands). Nucleic acid polymerases typically utilize a template polynucleotide (in either a single-strand or double-strand form) for nucleic acid synthesis, as in conventional nucleic acid replication, transcription, or reverse transcription. Other nucleic acid polymerases, e.g., terminal transferase (TdT), are capable of de novo polymerization, that is, template independent nucleic acid synthesis.
All known nucleic acid polymerases possess an enzymatic domain that catalyzes the formation of a phosphodiester bond between two nucleotides, utilizing the 5′ carbon triphosphate of one nucleotide and the 3′ carbon hydroxyl group of another nucleotide. Nucleic acid polymerases synthesize nascent polynucleotides by linking the 5′ phosphate of one nucleotide to the 3′ OH group of the growing polynucleotide strand. This process is known and commonly referred to by persons skilled in the art as 5′-3′ polymerization.
In addition, nucleic acid polymerases possess a wide range of ancillary chemical properties useful for nucleic acid synthesis. These properties include, but are not limited to:                product and/or template specificity (e.g., RNA or DNA);        single-strand or double-strand template specificity;        processivity—a measure of the ability of a nucleic acid polymerase to generate a nascent polynucleotide from a template polynucleotide without dissociating from the template;        extension rate—a measure of the rate at which nucleotides are added to a growing polynucleotide strand;        fidelity—a measure of the accuracy (or conversely the error rate) with which a nucleic acid polymerase synthesizes a polynucleotide complementary to a template polynucleotide;        nick translation—the ability of a nucleic acid polymerase to degrade the preceding nucleotide strand of a double strand molecule simultaneous to polymerizing a nascent strand;        proofreading—the ability of a nucleic acid polymerase to remove an incorrectly linked nucleotide from a polynucleotide before further polymerization occurs; and        thermostability—the ability of a nucleic acid polymerase to retain activity after exposure to elevated temperatures.        
Many of these properties are the result of one or more discrete functional domains within a polymerase holoenzyme. Three extensively studied enzymatically active domains of nucleic acid polymerase include: a 5′-3′ polymerase domain, responsible for polynucleotide synthesis; a 5′-3′ exonuclease domain, responsible for polynucleotide digestion of the 5′ end of a polynucleotide, useful for nick translation; and a 3′-5′ exonuclease domain, responsible for polynucleotide digestion of the 3′ end of a polynucleotide, allowing for proofreading, and thus improving the fidelity of the polymerase. Some studies indicate that selection, incorporation, and extension of the correct nucleotide, versus an incorrect nucleotide, is a variable property of the 5′-3′ polymerase domain, thus affecting polymerase fidelity in concert with proofreading activity (Mendelman et al., 1990; Petruska et al., 1988).
DNA polymerases can be categorized into six families based on amino acid homology. These families consist of; pol I, pol α,SONDZEICHEN pol β, SONDZEICHEN DNA-dependent RNA polymerase, reverse transcriptase, and RNA-dependent RNA polymerase (Joyce and Steitz, 1994). Table 1 summarizes the enzymatic features of a few representative DNA polymerases.
TABLE 1DNA polymerase enzymatic activity(N terminusC terminus)DNA5'-3'3'-5'5'-3'Therma-de novapolymeraseexanucleaseexonucleasepolymerasestabilitypolymeraseE. coli pol I (+)(+)(+)(−)(−)Klenaw(−)(+)(+)(−)(−)fragmentE. coli pol II(−)(+)(+)(−)(−)E. coli pol III(+)(+)(+)(−)(−)T4 pol(−)(+)(+)(−)(−)T7 pol(−)(+)(+)(−)(−)M-MuLV RT(−)(−)(+)(−)(−)TdT(−)(−)(+)(+)(+)Taq pol(+)(−)(+)(+)(−)Stoffel(−)(−)(+)(+)(−)fragmentTbr pol(+)(−)(+)(+)(−)Tli pol(−)(+)(+)(+)(−)Tma pol(−)(+)(+)(+)(−)Tth pol(+)(−)(+)(+)(−)Pfu pol(−)(+)(+)(+)(−)Psp pol(−)(+)(+)(+)(−)Pwo pol(−)(+)(+)(+)(−)
Because of the diversity of properties and characteristics potentially exhibited by nucleic acid polymerases generally, practitioners in the art have sought to modify, to alter, or to recombine various features of nucleic acid polymerases in an effort to develop new and useful variants of the enzyme. Initially, polymerase truncations and deletions were developed. The Klenow fragment, for example, was the first nucleic acid polymerase variant developed. Klenow fragments exist as a large C-terminal truncation of DNA polymerase I (pol I), possessing an enzymatically active 3′-5′ exonuclease and 5′-3′ polymerase domains, but lacking altogether the 5′-3′ exonuclease domain of native pol I (Klenow and Henningsen, 1970; Jacobson et al., 1974; and Joyce and Grindley, 1983).
Since the advent of the polymerase chain reaction (PCR) methodology (including derivative methodologies such as reverse transcription PCR, or RT-PCR), resilient nucleic acid polymerases, capable of withstanding temperature spikes as high as 95° C. without a subsequent significant loss in enzymatic activity (i.e., thermostable) have become vital tools in modern molecular biology. The use of thermostable enzymes to amplify nucleic acid sequences is described in U.S. Pat. Nos. 4,683,195 and 4,683,202. A thermostable DNA polymerase from Thermus aquaticus (Taq) has been cloned, expressed and purified from recombinant cells (Lawyer et al., 1989; U.S. Pat. Nos. 4,889,818 and 5,079,352. PCR is also described in many U.S. patents, including U.S. Pat. Nos. 4,965,188, 4,683,195, 4,683,202, 4,800,159, 4,965,188, 4,889,818, 5,075,216, 5,079,352, 5,104,792, 5,023,171, 5,091,310, and 5,066,584.).
As depicted in Table I, Taq DNA polymerase possesses enzymatically active 5′-3′ polymerase and 5′-3′ exonuclease domains, but it exhibits only background levels of 3′-5′ exonuclease activity (Lawyer et al., 1989; Bernard et al., 1989; Longley et al., 1990). Crystallographic data revealed that Taq polymerase contains a 3′-5′ exonuclease domain (Eom et al., 1996); comparisons of the crystal structure of the Klenow fragment from Bacillus DNA polymerase I, Taq DNA polymerase, and E. coli DNA polymerase I have shown, however, that critical residues required to carry out a 3′-5′ exonuclease activity are missing in the 3′-5′ exonuclease domain of Taq DNA polymerase (Kiefer et al., 1997). Park et al. (1997), have determined that Taq DNA polymerase possesses none of three sequence motifs (Exo I, II, and III) within the 3′-5′ exonuclease domain and necessary for 3′-5′ exonuclease activity. Because Taq polymerase exhibits essentially no 3′-5′ exonuclease activity (i.e., proofreading capability), the error rate of Taq DNA polymerase is high compared to other DNA polymerases that possess an enzymatically active 3′-5′ exonuclease domain (Flaman et al., 1994). The Taq DNA polymerase structure thus comprises a 5′-3′ exonuclease domain occurring at the N-terminal region of the polypeptide (residues 1-291), followed by an enzymatically inactive 3′-5′ exonuclease domain (residues 292-423), and a C-terminal 5′-3′ polymerase domain (Park et al., 1997).
Since Taq DNA polymerase does not possess an enzymatically active 3′-5′ exonuclease domain, providing a proofreading feature to the polymerase, the use of Taq DNA polymerase becomes less desirable for most nucleic acid amplification applications, e.g., for PCR sequencing protocols or amplification for protein expression, which require complete identity of replication products to the template nucleic acid. Depending on the phase of PCR during which an error becomes incorporated into the PCR product (e.g., in an early replication cycle), the entire population of amplified DNA could contain one or more sequence errors, giving rise to a nonfunctional and/or mutant gene product. Nucleic acid polymerases that possess an enzymatically active 3′-5′ exonuclease domain (i.e., proofreading activity), therefore, are especially preferred for replication procedures requiring high fidelity.
Due to the scientific and commercial importance of PCR in modern molecular biology, the reliance of PCR protocols on nucleic acid polymerases of particular characteristics, and in view of the enzymatic deficiencies of Taq polymerase, an enormous amount of research and development has focussed on developing new and useful thermostable DNA polymerase variants and/or assemblages.
One approach has been directed to the discovery and isolation of new thermophilic nucleic acid polymerases, which may possess a unique and/or improved collection of catalytic properties. As a result, thermostable nucleic acid polymerases have been isolated from a variety of biological sources, including, but not limited to, species of the taxonomic genera, Thermus, Thermococcus, Thermotoga, Pyrococcus, and Sulfolobus. These polymerases possess a variety of chemical characteristics, as illustrated in Table 1. Some of these naturally occurring thermostable DNA polymerases possess enzymatically active 3′-5′ exonuclease domains, providing a natural proofreading capability and, thus, exhibiting higher fidelity than Taq DNA polymerase. Naturally occurring proofreading thermostable polymerases include: Pfu polymerase (isolated from Pyrococcus furiosus), Pwo polymerase (isolated from Pyrococcus woesei), Tli polymerase (isolated from Thermococcus litoralis), and Psp polymerase (isolated from Pyrococcus sp. GB-D). All of these naturally occurring thermostable polymerases are commercially available (Tli polymerase and Psp polymerase are marketed as Vent® and Deep VentSONDZEICHEN® DNA polymerase, respectively, by New England Biolabs, Beverly, Mass.). These DNA polymerases show slower DNA extension rates and an overall lower processivity when compared to Taq DNA polymerase, however, thus rendering these naturally occurring thermostable DNA polymerases less desirable for PCR, despite their higher fidelity.
In an effort to compensate for the deficiencies of individual thermostable polymerases, a second approach has been to develop multiple enzyme assemblages, combining, for example, Taq polymerase and a proofreading enzyme, such as Pfu polymerase or VentSONDZEICHEN® polymerase. These multiple-enzyme mixtures exhibit higher PCR efficiency and reduced error rates when compared to Taq polymerase alone (Barnes, 1994). Mixtures of multiple thermostable enzymes are commercially available (e.g., the Failsafe™ PCR system from Epicentre, Madison, Wis.). PCR protocols utilizing multiple polymerase mixtures are still prone to error, however, and require the practitioner to perform preliminary experimental trials, to determine special optimized solution conditions necessary for multiple-enzyme reaction mixtures.
A third approach has been to develop new and useful variants of Taq polymerase through deletion/truncation techniques. The Stoffel fragment, for example, is a 544 amino acid C-terminal truncation of Taq DNA polymerase, possessing an enzymatically active 5′-3′ polymerase domain but lacking 3′-5′ exonuclease and 5′-3′ exonuclease activity. Other commercially available thermostable polymerase deletions include VentSONDZEICHEN® (exo−) and Deep VentSONDZEICHEN® (exo−) (New England Biolabs, Beverly, Mass.). Deletion mutations serve only to remove functional domains of a nucleic acid polymerase, however, and do not add any novel features or enzymatic properties.
Polymerase mutagenesis is yet another approach that has been attempted to develop new and useful nucleic acid polymerase variants. Park et al. (1997) performed site-directed mutagenesis of 4 amino acids in the enzymatically inactive 3′-5′ exonuclease domain of Taq polymerase in an effort to activate the proofreading ability of this domain. The resultant mutant exhibited an increase of exonuclease activity over that of naturally occurring Taq polymerase. The reported increase was a mere two-fold increase above background exonuclease activity, however; an insignificant rise in exonuclease activity that is unlikely to increase PCR fidelity.
Bedford et al. (1997) developed a recombinant mesophilic DNA pol I from E. coli. They succeeded to insert a thioredoxin binding domain from T7 DNA polymerase into E. coli pol I. The inserted 76 amino acid binding domain improved polymerase binding to a template polynucleotide, thus increasing the processivity of the recombinant E. coli pol I but did not improve or provide any novel enzymatic activity to the polymerase.
Recently Gelfand et al. (1999) combined fusion protein technology with mutagenesis to eliminate or substantially reduce 5′-3′ exonuclease activity and 3′-5′ exonuclease activity in recombinant polymerases. Once again, no improved or additional enzymatic activity was provided by the fusion polymerase.
Frey et al. (1999) attempted to engineer chimeric polymerases utilizing enzymatically active domains from Taq, Tne, and E. coli DNA polymerases. Although they successfully substituted the non-functional 3′-5′ exonuclease domain of Taq DNA polymerase with a functional 3′-5′ exonuclease domain from another DNA polymerase, their resultant chimeric polymerase lost significant, if not all, enzymatic activity after only one minute at 80° C. or 95° C. (i.e., they are not thermostable), and thus are not useful for performing PCR protocols without the successive addition of fresh polymerase for each cycle.
Despite these intense research efforts, there remains a need in the art for thermostable nucleic acid polymerases that possess improved or novel assemblages of enzymatically active domains. Despite its enzymatic deficiencies, Taq DNA polymerase remains the most widely used enzyme for processing in vitro amplification of nucleic acids. In particular, there has been long felt need for a nucleic acid polymerase possessing the 5′-3′ polymerization qualities of Taq polymerase, but which also possesses 3′-5′ exonuclease (proofreading) activity.